The use of antisense oligonucleotides offers advantages over other therapeutic regimes due to their potential for target specificity. For example, conventional chemotherapy for neoplastic and virus-related diseases has the disadvantage of systemic toxicity. The therapeutic index for chemotherapeutic agents is relatively narrow, since such agents are unable to distinguish between normal and diseased cells. Antisense oligonucleotides have the potential to be many orders of magnitude more specific than traditional drugs due to their greater number of interactions with a particular target site. In theory, an oligonucleotide of more than 15-17 nucleotides in length could have the base pairing specificity to interact with only one target gene within the entire human genome. Thus, antisense oligonucleotides have the potential specificity that could serve as a powerful tool for the study of specific gene function and as therapeutic agents for disease-causing genes.
In contrast to drugs, antisense molecules are relatively simple to design. The interaction between an antisense oligonucleotide and a target mRNA is governed primarily by the sequence of the target. Oligonucleotides targeting the start codon and extending upstream or downstream have been shown to be effective. Similarly, oligonucleotides that are complementary to the splice sites have proved effective.
Antisense technologies for the targeted inhibition of gene expression could provide an effective strategy for the management of inherited disorders with dominant-negative or gain-of-function pathogenetic mechanisms, for the suppression of oncogenes, or for the control of a variety of infectious agents. Pathologic disorders that are currently targeted by antisense therapeutics include viral infections, inflammatory disorders, cardiovascular disease, cancers, genetic disorders and autoimmune diseases. Synthetic oligodeoxynucleotides (ODNs), especially phosphorothiates and methylphosphonates, offer the advantage of enhanced stability in biological fluids and an effectively limitless supply.
Antisense oligonucleotides are also useful for the production of transgenic animals having alterations at the germline level, such as knockout mutations, which can be used for the study of new genes or the study of the function of a known gene. Further, antisense technology combined with gene therapy is usefull for example, for suppression of expression of a mutant gene product. Such gene therapy would be most advantageous in combination with a replacement regimen utilizing the “normal” gene to provide a “normal” gene product.
Unfortunately, the effective use of antisense oligonucleotides has been limited due to several problems. Disadvantages include the transient nature of ODNs, and their toxicity and propensity for producing non-sequence specified biological effects. Other disadvantages include low expression or limited stability of complementary RNAs which result in their nonspecific targeting or low efficiency of target inhibition. Antisense oligonucleotides are often poorly taken up by cells and therefore may never reach their target site. Often, antisense oligonucleotides do not reach the nucleus of a cell once administered, the site of their RNA and DNA targets. In certain applications the antisense molecules are microinjected directly into the cells. This technique works well in the laboratory, however, it cannot be applied to patients. Many of the studies with antisense show that gene expression is suppressed by 80-90% of the normal level, however, such reduction is not typically sufficient to reduce the biological effect, e.g., 10-20% expression is sufficient to maintain the biological function sought to suppress.
There is a need to develop a delivery system for antisense molecules that gives the antisense enhanced stability, for example by being resistant to nuclease activity or by being enriched in the nucleus, while still allowing specificity of the antisense for its target RNA or DNA. Such a system would provide effective targeting of the message with the end result being significant inhibition of expression of a particular gene.